Saxena Shivalika, Shukla Surendra, Thakur Akhilesh, Gupta Reena
Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171 005, India.
Acta Microbiol Immunol Hung. 2008 Mar;55(1):33-51. doi: 10.1556/AMicr.55.2008.1.3.
The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.
目前的工作重点是将聚半乳糖醛酸酶高效固定在聚乙烯基质上，随后将其应用于苹果汁澄清。迄今为止，尚未见聚半乳糖醛酸酶在活化聚乙烯上的固定化及其在苹果汁澄清中的应用报道。黑曲霉（MTCC 3323）在含有果胶作为单一碳源的改良里维耶培养基中通过分批补料培养产生聚半乳糖醛酸酶。该酶用乙醇沉淀，通过凝胶过滤色谱法（Sephacryl S - 100）纯化，并固定在戊二醛活化的聚乙烯上。该方法用于酶固定化非常简单且省时。与游离酶相比，评估了固定化酶的各种特性，如最佳反应温度和pH、温度和pH稳定性、结合动力学、结合效率、可重复使用性以及金属离子对固定化酶的影响。游离酶和固定化酶在45℃温度和pH 4.8时均表现出最大活性。最大结合效率为38%。固定化酶可重复使用3个循环，第三个循环后活性损失50%。在45℃和孵育1小时条件下，24 U固定化酶使苹果汁在650 nm处的透光率提高了约55%。就坚固性、可用性、惰性、低价格、可重复使用性和温度稳定性而言，固定化酶具有工业优势。
